Presenting Author Middle Tennessee State University Murfreesboro, Tennessee
IU-NH catalyzes the hydrolysis of selected nucleosides to the corresponding base and pentose sugar. This enzyme plays a central role in the salvage pathway of nucleosides. Kinetic studies with inosine and uridine along with other analogs of these nucleosides were used to characterize the substrate specificity of the enzyme. Uridine was found to be the preferred substrate over inosine with a KM of 2,099 μM compared to 509 μM. The enzyme was found to have broad specificity. Substitution at sites in the nucleoside substrate were used to determine the role these sites played in the catalytic mechanism. Site-directed mutagenesis studies on selected amino acid residues were used to determine the role these residues play in the binding of the nucleoside to the enzyme and the catalytic mechanism. Molecular dynamic studies in conjunction with the experimental determination of substrate specificity were used to build a visual picture of the catalytic mechanism. A flexible loop around the active site acts in a pincer motion to maintain and stabilize the bound nucleoside conformation and cleave the glycosidic bond. Calcium is suspected to play a central role in how the flexible loop interacts with residues of the active site by using calcium to complete a hydrolytic union between water, C1’ of the nucleoside, and N9 of a purine base or N1 of a pyrimidine base. The mechanism of the enzyme isolated from Arabidopsis was compared to the well-characterized enzyme isolated from parasitic protozoans.
Support or Funding Information
MTSU Department of Chemistry MTSU Molecular Bioscience Program
MTSU Department of Chemistry MTSU Molecular Bioscience Program
